The effects of freezing on faecal microbiota as determined using MiSeq sequencing and culture-based investigations.
The effects of freezing on faecal microbiota as determined using MiSeq sequencing and culture-based investigations.
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DOI：
10.1371/journal.pone.0119355
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Stanton C
中科院分区：
文献类型：
--
作者：
Fouhy F;Deane J;Rea MC;O'Sullivan Ó;Ross RP;O'Callaghan G;Plant BJ;Stanton C
高通量测序使人们能够详细了解复杂的微生物环境，包括人类肠道微生物区系。然而，测序数据的准确性取决于DNA提取前样品的适当存储。这项研究的目的是对粪便储存对微生物区系的影响进行第一次MiSeq测序调查，并与新鲜样本进行比较。还完成了基于文化的分析。从健康成人身上采集了7份粪便样本。将样品分成新鲜的(立即提取DNA)，在干冰上快速冷冻，并在-80°C下冷冻7天后提取DNA，或在-80°C下冷冻7天后提取DNA。测序在Illumina MiSeq平台上完成。完成了总需氧菌、厌氧菌和双歧杆菌的培养。治疗组之间在门类或家庭水平上没有显著差异。在属水平上，新鲜样品与速冻样品有显著差异(p=0.0298；p=0.0330)。多样性分析表明，样本是根据个人捐赠者而不是按储存组分组的。新鲜样品、速冻样品和-80°C冷冻样品之间的培养分析没有显著差异。利用MiSeq平台和基于培养的分析，这项研究强调，在提取DNA之前快速冷冻粪便样本后，微生物区系发生的显著变化有限。因此，在提取和培养DNA之前快速冷冻样品，可以保持微生物区系的完整性。
High-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed. Seven faecal samples were collected from healthy adults. Samples were separated into fresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°C prior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencing was completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobes and bifidobacteria was also completed. No significant differences at phylum or family levels between the treatment groups occurred. At genus level only Faecalibacterium and Leuconostoc were significantly different in the fresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversity analysis indicated that samples clustered based on the individual donor, rather than by storage group. No significant differences occurred in the culture-based analysis between the fresh, snap or -80°C frozen samples. Using the MiSeq platform coupled with culture-based analysis, this study highlighted that limited significant changes in microbiota occur following rapid freezing of faecal samples prior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing, preserves the integrity of the microbiota.
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影响因子：
4.2
作者：
Wu GD;Lewis JD;Hoffmann C;Chen YY;Knight R;Bittinger K;Hwang J;Chen J;Berkowsky R;Nessel L;Li H;Bushman FD
通讯作者：
Bushman FD
DOI：
10.2174/1874285800903010040
发表时间：
2009
期刊：
The open microbiology journal
影响因子：
--
作者：
Roesch LF;Casella G;Simell O;Krischer J;Wasserfall CH;Schatz D;Atkinson MA;Neu J;Triplett EW
通讯作者：
Triplett EW
影响因子：
3.2
作者：
Simpson, PJ;Stanton, C;Ross, RP
通讯作者：
Ross, RP
影响因子：
2.1
作者：
Bahl, Martin Iain;Bergstrom, Anders;Licht, Tine Rask
通讯作者：
Licht, Tine Rask
影响因子：
2.1
作者：
Lauber CL;Zhou N;Gordon JI;Knight R;Fierer N
通讯作者：
Fierer N